Hydrogen sulfide (H2S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions [1-5]. It has also become the focus of many research endeavors, including pharmaco-therapeutic manipulation [1-5]. One of the challenges facing the field is the accurate measurement of biologically active H2S. The complexity of analytical H2S measurement, especially in living organisms, reflects the fact that hydrogen sulfide is a volatile gas and exists in the organism in different forms, including a free form (“free H2S”), an acid labile pool, and as bound sulfane sulfur, as shown in FIG. 1.
Sulfur exists in the body in several forms, ranging from a fully reduced divalent state as sulfide to a fully oxidized hexavalent state as sulfate [1, 9, 10]. Measurement of biologic sulfur has focused on measuring sulfide (the reduced divalent state), in part because of difficulties in accurately measuring other states. Sulfur equivalents in the reduced divalent state are very reactive within biological matrices, resulting in sulfide equivalents being present in three different volatile sulfur pools, as shown in FIG. 1. All three pools are important in regulating the amount of bioavailable sulfur with the most important being the acid labile and bound sulfane sulfur pools [10, 11].
Hydrogen sulfide is produced predominantly enzymatically from cysteine, for example, using two pyridoxal-5′-phosphate dependent enzymes, cystathionine-β-synthase and cystathionine-γ-lyase, as well as 3-mercaptosulfurtransferase. Free hydrogen sulfide can diffuse across cellular membranes without the need for a specialized transporter [4, 6]. Free H2S is found dissolved in plasma and other tissue fluids. At mammalian body conditions, i.e., pH 7.4 and temperature of 37° C., 18.5% of free hydrogen sulfide exists as H2S gas, and the remainder is almost all hydrosulfide anion (HS—) with a negligible contribution of S2− [7, 8].
Sulfane sulfur refers to divalent sulfur atoms bound to another sulfur, though they may bear an ionizable hydrogen at some pH values. Examples of these bound sulfurs include thiosulfate S2O32−, persulfides R—S—SH, thiosulfonates R—S(O)—S—R′, polysulfides R—Sn—R, polythionates SnO62−, and elemental sulfur S0 [10]. These sulfane-bound sulfurs can be released under reducing conditions. Acid labile sulfide, the other major bioavailable pool, consists of sulfur present in iron-sulfur clusters contained in iron-sulfur proteins (non-heme), which are ubiquitous in living organisms, and include a variety of proteins and enzymes, including without limitation, rubredoxins, ferredoxins, aconitase, and succinate dehydrogenase [10, 12]. The acid labile sulfides readily liberate free H2S in acid conditions (pH<5.4). The process of acid liberation may also release hydrogen sulfide from persulfides, which have traditionally been classified as sulfane sulfur [13]. This “acid labile sulfide pool” has been postulated to be a “reversible sulfide sink” and may be an important storage pool that regulates the amount of bioavailable free hydrogen sulfide [14]. However, the bound sulfur forms may be more important in storing and release of exogenously administered sulfide [11].
A weakness to the study of sulfide has been the lack of precise methodology for the accurate and reproducible measurement of hydrogen sulfide both in vivo and in vitro. A variety of methods to measure free H2S have been employed with divergent results [10, 13, 15]. These methods include a spectrophotometric derivatization method resulting in methylene blue formation, variations of this methylene blue method using high performance liquid chromatography [10], sulfide ion-selective electrodes, polarographic sensors [16], gas chromatography [13, 17], and HPLC in conjunction with fluorimetric based methods using monobromobimane (MBB) to derivatize free H2S [14, 18, 39, 41].
The levels of H2S in a mammalian body that have been measured range from nanomolar to hundreds of micromolar concentrations [10, 15]. This wide range is partially due to the various methods of measuring the H2S. The previously favored methylene blue method of hydrogen sulfide detection had several disadvantages: the method had interference from bound sulfide pools, was subject to chemical artifacts, and was unable to actual free hydrogen sulfide. Moreover, methylene blue readily forms dimer and trimer aggregates in aqueous media that does not conform to Beer's law which further prevents accurate analytical measurement of bio available sulfide [18].
Earlier attempts to characterize the bound sulfane sulfur pool have primarily utilized MBB in conjunction with dithiothreitol (DTT) as a reducing agent to free the bound sulfide [10, 21]. Most work has focused on the free hydrogen sulfide and acid labile pools alone [11, 13]. These study results were limited because of various problematic issues such as pH, volatilization, and oxidation of the measured samples [40].
The fluorescent reagent MBB has been widely used to measure various thiol-containing species through alkylation [22]. S-alkylation occurs twice with sulfide under alkaline conditions, forming sulfide-dibimane. We have previously reported a fluorimetric, reverse-phase (RP)-HPLC analytical method that stabilizes biologically active free hydrogen sulfide from oxidation while able to detect low levels. This analytical method measured free plasma hydrogen sulfide by derivatization of sulfide with an excess of MBB under alkaline, low oxygen, and trace metal-free conditions with RP-HPLC separation and fluorescent detection of the fluorescent sulfide-dibimane product with a detection level of about 2 nM [18].
The field of hydrogen sulfide measurement continues to evolve with modifications of various methods, including the report of different fluorescent probes [33-35] as well as applications of new technologies, such as nanotubes and quantum dots [36, 37], and a method for measurement of hydrogen sulfide dissolved in aqueous solutions through the use of an electrochemical sensor [38]. However, there is no reported method for accurately measuring all labile hydrogen sulfide pools to determine hydrogen sulfide bioavailability in biological or other samples that contain biologically derived tissues or proteins, e.g., environmental water samples. There is a need for a method that allows for an accurate, quantitative, and scalable measurement of discrete pools of hydrogen sulfide from primary labile sulfide pools to use in both experimental and clinical samples.
U.S. Pat. No. 6,468,762 discloses a method to measure homocysteine using N,N-dipropyl-phenylene diamine and using dithiothreitol as a reducing agent.
U.S. Patent Application Publication No. 2007/0078113 discloses a method to measure hydrogen sulfide in blood using an extractive alkylation technique coupled with gas chromatography and mass specific detection to quantify hydrogen sulfide, and using a denaturing/reducing reaction buffer of benzalkonium chloride, tetraethylammonium hydroxide, and tris(2-carboxyethyl)phosphine hydrochloride in saturated borate buffer.
U.S. Patent Application Publication No. 2012/0073988 discloses an electrochemical sensor to measure hydrogen sulfide.